Bone morphogenetic proteins (BMPs) are growth factors that control the development and maintenance of most tissues by regulating cell proliferation, differentiation and apoptosis. BMPs also stimulate bone repair and augmentation in post-natal animals. Because of the bone forming capacity of BMPs in several animal in vivo and in vitro models, BMPs have received much attention as bone therapeutic agents. However, human clinical trials show show a relatively poor BMP response and large patient-to-patient variabilities. The osteogenic response to BMPs of adult human bone-marrow derived mesenchymal stem cell (hMSC) cultures is charactarized by poor induction of the early osteoblast genes alkaline phosphatase and osteopontin, although BMPs efficiently induce these genes in rat or mouse MSC cultures. Our general hypothesis is that BMP-mediated osteogenesis of hMSC in vitro is negatively regulated by elevated ERK activity induced by serum, and positively regulated by p38 and PI3-K/AKT signaling. In this application we propose to investigate mechanisms by which kinase pathways regulate BMP-mediated in vitro osteogenesis of hMSC and elucidate whether BMP-activated Smads operate in conjuction with kinase pathways to induce early osteoblast genes. Aim1 will test the hypothesis that serum-activated ERK phosphorylates Smads, thereby reducing BMP activation of Smads, Smad nuclear localization and Smad transcriptional activity. Aim2 will tests the hypothesis that BMP-induced early osteogenic genes are direct targets of p38 and PI3-K/ AKT signaling, rather than direct Smad signaling. The knowledge gained from the present study will provide new insights into mechanisms of BMP osteogenic action in human MSC and form the basis for more extensive examination of BMP-regulated osteogenesis in humans. Our long-term goal is to develop clinically improved bone therapies with BMPs and human MSC. [unreadable] [unreadable]